Purification and characterization of proteinase inhibitors from winged bean (Psophocarpus tetragonolobus (L.) DC.) seeds

Seven proteinase inhibitors were isolated from winged bean seeds by ion-exchange chromatographies. These inhibitors had molecular weights of around 20,000, included four half-cystine residues, and were Kunitz-type inhibitors. Two (WTI-2 and 3) inhibited bovine trypsin strongly and four (WCI-1, 2, 3, and 4) inhibited bovine alpha-chymotrypsin, but in different ways. One mole of WCI-2 or -3 could inhibit 2 mol of alpha-chymotrypsin. The remaining inhibitor (WTCI-1) could bind both bovine trypsin and alpha-chymotrypsin at the molar ratio of 1:1, but not simultaneously. All four chymotrypsin inhibitors cross-reacted with rabbit anti-WCI-3 serum, while the other inhibitors did not.     

Two Isolectins from Leaves of Winged Bean, Psophocarpus tetragonolobus (L.) DC.

Two isolectins were isolated from leaves of winged bean andcharacterized. They differed from each other in terms of theirimmunological properties, hemagglutinating activities, sugarinhibition patterns, and amino acid compositions. Both lectinswere acidic and one of them (L-I), which was inactive towardtrypsinized human type O erythrocytes, was similar to one ofgreen shell lectins (WGS-1); which resembled basic seed lectinin its immunological properties. The amino-terminal sequenceof L-I was homologous to that of WGS-1. The amino acid compositionof L-I was similar to that of basic seed lectin, but the extentof the homology between amino-terminal sequences was low whenL-I and basic seed lectin were compared. Examination by ELISArevealed that L-I and WGS-1 were distinct from the basic lectinsof seeds and tuberous roots. L-I had a disulfide bridge betweentwo subunits and it exhibited high hemagglutinating activitytoward human type A erythrocytes, as compared to its activitytoward other erythrocytes. By contrast, the properties of asecond acidic lectin from winged bean leaves (L-II) were verysimilar to those of acidic lectins from seeds and tuberous roots,and the similarities extended as far as the immunological properties. (Received January 6, 1994; Accepted August 15, 1994)     

Characterization of the acidic lectins from winged bean seed (Psophocarpus tetragonolobus(L.)DC)

The seeds of winged bean, Psophocarpus tetragonolobus(L.)DC, contain two distinct groups of lectins characterized by different erythrocyte hemagglutinating specificities and isoelectric points. Three acidic lectins (I, II, and III) (pI approximately 5.5) were purified to apparent homogeneity by chromatography on Ultrogel AcA44 and SP-Sephadex C-25. These lectins are glycoproteins with relative molecular mass of 54,000. The total carbohydrate content of the acidic lectins was 7% and was comprised of mannose, N-acetylglucosamine, fucose, and xylose in amounts corresponding to 9.2, 4.8, 1.6, and 7.0 mol/54,000 g, respectively. Electrophoresis in dodecyl sulfate, in the presence and absence of 2-mercaptoethanol, gave a single subunit of apparent relative molecular mass 30-32,000, somewhat higher than expected from the native relative molecular mass. On isoelectric focusing in 8 M urea the subunits of the acidic lectins did not show any significant charge heterogeneity as found for the winged bean basic lectins. The acidic lectins have very similar amino acid compositions. They contain essentially no half-cystine, 1-2 methionine residues, and are rich in acidic and hydroxy amino acids. The amino-terminal sequences of lectins II and III were identical while the amino-terminal sequence of lectin I contained five differences in the first 25 residues; the acidic lectins showed extensive sequence homology with the winged bean basic lectins, the other one-chain subunit lectins and the beta subunit of the two-chain subunit legume lectins. The acidic lectins agglutinated trypsinized human (type A, B, AB, and O) erythrocytes but not trypsinized rabbit erythrocytes. They were inhibited by various D-galactose derivatives and D-galactose-containing disaccharides and trisaccharides. N-Acetylgalactosamine was the best inhibitor, and the specificity appears to be directed to beta-D-galactosides. However, compared with winged bean basic lectins and soybean lectin, the winged bean acidic lectins show a low affinity for the inhibitory sugars.     

Conformation and stability of the chymotrypsin inhibitor from winged bean seed (Psophocarpus tetragonolobus (L.) DC)

Spectrophotometric measurement was found to be a sensitive method for evaluating the stability of the chymotrypsin inhibitor from the winged bean. The thermal stability of this protein in aqueous solution was much greater at pH 3 than at pH 8 or pH 11. Evidence from u.v. absorption and from circular dichroism indicated that irreversible conformation changes occurred at higher temperature (greater than 70 degrees). Circular dichroism and optical rotatory dispersion studies at pH 8 show that the inhibitor is rich in beta-structure and virtually devoid of alpha-helix in aqueous solution. We conclude from experiments with denaturing solvents that the inhibitor is very stable and that high concentrations of denaturant are required before unfolding occurs. Chemical modification experiments with tetranitromethane were consistent with a tight stable structure; even in 6M guanidine hydrochloride only three of the five tyrosine residues in the inhibitor molecule were nitrated. However, tyrosine does not seem to be implicated at the reactive site of the inhibitor. Interaction of the inhibitor with alpha-chymotrypsin and chymotrypsin B was also followed by difference spectroscopy in the ultraviolet region. Difference spectra were detected that were characteristic of changes in the environment of both tyrosine and tryptophan chromophores. Comparison of the spectral data obtained for the interaction of the inhibitor with bovine alpha-chymotrypsin and with chymotrypsin B indicated that a tryptophan residue may be involved at the reactive site of the inhibitor. Spectral changes were also detected for the interaction between the chymotrypsin inhibitor and trypsin, although it is well established that the specificity of this inhibitor is restricted to the chymotrypsins.(ABSTRACT TRUNCATED AT 250 WORDS)     

Lectins in Immature Pods of Winged Bean,Psophocarpus tetragonolobus (L.) DC.

Lectin activity in immature pods from 30 strains of winged bean was investigated. Most of the lectin activity occurred in green shells and a small portion in immature seeds. Hemagglutinating activity of green shells was classified into four groups, according to the agglutination of trypsinized human type A, B, and O erythrocytes. Extracts from green shells of four representative strains which showed different hemagglutination patterns gave different elution profiles of lectin activity from ion exchange columns. Four types of lectin activity with different blood group specificities were apparently found in green shells.     

Amino acid sequences of two trypsin inhibitors from winged bean seeds (Psophocarpus tetragonolobus (L)DC.)

The trypsin inhibitor (WTI-1) purified from winged bean seeds is a Kunitz type protease inhibitor having a molecular weight of 19,200. WTI-1 inhibits bovine trypsin stoichiometrically, but not bovine alpha-chymotrypsin. The approximate Ki value for the trypsin-inhibitor complex is 2.5 X 10(-9) M. The complete amino acid sequence of WTI-1 was determined by conventional methods. Comparison of the sequence with that of soybean trypsin inhibitor (STI) indicated that the sequence of WTI-1 had 50% homology with that of STI. WTI-1 was separated into 2 homologous inhibitors, WTI-1A and WTI-1B, by isoelectric focusing. The isoelectric points of WTI-1A and WTI-1B were 8.5 and 9.4, respectively, and their sequences were presumed from their amino acid compositions.     

Sequence and expression of the mRNA encoding the chymotrypsin inhibitor in winged bean (Psophocarpus tetragonolobus (L.) DC.)

The accumulation of the Kunitz-type chymotrypsin inhibitor WCI-3 in winged bean seeds is controlled developmentally. In vitro translation experiments showed that the WCI-3 mRNA was present in 35- and 40-day-old immature seeds after flowering. The size of the in vitro translation product is about 2 000 Da larger than that of the mature WCI-3 protein. The WCI-3 cDNA clones were isolated from a gtll cDNA library of 35-day-old immature seeds by immunoscreening. A nearly full-length cDNA clone was obtained containing an open reading frame of 207 amino acid residues. The deduced sequence of the 183 carboxy terminal amino acids coincides precisely with the amino acid sequence determined for purified WCI-3. The amino terminal extension of 24 residues has the characteristics of a signal peptide. Northern hybridization analysis of total poly(A)⁺ RNA showed that the WCI-3 mRNA is approximately 900 nucleotides long and accumulates in 35- and 40-day-old but not in 30-day-old immature seeds.     

Direct somatic embryogenesis and plantlet regeneration from seedling leaves of winged bean,Psophocarpus tetragonolobus (L.) DC

Excised seedling leaf segments of winged bean [Psophocarpus tetragonolobus (L.) DC.] underwent direct somatic embryogenesis under appropriate incubation conditions. Initiation and development of the somatic embryos occurred using a two-step culture method. The culture procedure involved incubation for 28 days on MS basal medium supplemented with 0.1–0.5 mg/l NAA and 1.0–2.0 mg/l BA (induction medium) before transfer to MS medium supplemented with 0.1 mg/l IAA and 2.0 mg/l BA (embryo development medium). The initial exposure to low levels of NAA coincident with high levels of BA in the induction medium was essential for embryogenic induction. Maximum embryogenesis (43.3%) was obtained with 0.2 mg/l NAA and 2.0 mg/l BA, and at least 14 days on induction medium were required prior to transfer to the embryo development medium. The conversion frequency of cotyledonary embryos was 53.3% upon culture on MS medium containing 0.1 mg/l ABA for 7 days followed by transfer to MS medium supplemented with 0.1 mg/l IBA and 0.2 mg/l BA. Following conversion, the regenerated plantlets were transferred to soil and showed normal morphological characteristics.Abbreviations MS Murashige and Skoog (1962) medium - 2,4-D 2,4-dichlorophenoxyacetic acid - NAA 1-naphthaleneacetic acid - IAA indole-3-acetic acid - IBA indole-3-butyric acid - BA 6-benzylaminopurine - ABA abscisic acid     

Size and compositional heterogeneity of seeds in the winged bean pod (Psophocarpus tetragonolobus (L.)DC)

Differently positioned seeds in the mature pod of winged bean (Psophocarpus tetragonolobus (L.) DC) differ in mass and content of total protein and phosphorus and starch.     

Chemical modification studies on a lectin from winged-bean [Psophocarpus tetragonolobus (L.) DC] tubers.

The effect of chemical modification on a D(+)-galactose-specific lectin isolated from winged-bean tubers was investigated to identify the type of amino acid involved in its haemagglutinating activity. Various anhydrides of dicarboxylic acids, such as acetic anhydride, succinic anhydride, maleic anhydride and citraconic anhydride, modified 57-68% of the amino groups of the winged-bean tuber lectin. Treatment with N-acetylimidazole modified only 45% of the total amino groups. Reductive methylation of free amino groups modified 57% of the amino groups. Modification of the amino groups of the lectin by acetic anhydride and succinic anhydride did not lead to any significant change in the haemagglutinating activity (greater than or equal to 75% active). However, citraconylation and maleylation of the lectin led to a significant decrease in the haemagglutinating activity (less than or equal to 20% active). Acetylation and succinylation (3-carboxypropionylation) of the lectin led to a decrease in the pI value of the native lectin from approx. 9.5 to approx. 4.5. Treatment of the lectin with N-bromosuccinimide led to the modification of two and four tryptophan residues per molecule in the absence and in the presence of 8 M-urea respectively. The immunological identity of all the modified lectin preparations showed no gross structural changes except the lectin modified with N-bromosuccinimide in the presence of urea at pH 4.0.     

Amino acid sequence of winged bean (Psophocarpus tetragonolobus (L.) DC.) chymotrypsin inhibitor, WCI-3

The complete amino acid sequence of winged bean chymotrypsin inhibitor 3 (WCI-3) was determined by the conventional methods. WCI-3 consisted of 183 amino acid residues, but was heterogeneous in the carboxyl terminal region owing to the loss of one to four carboxyl terminal amino acid residues. The sequence of WCI-3 was highly homologous with those of soybean trypsin inhibitor Tia, winged bean trypsin inhibitor WTI-1, and Erythrina latissima trypsin inhibitor DE-3. One of the reactive site peptide bonds of WCI-3 was identified as Leu(65)-Ser(66), which was located at the same position as those of the other Kunitz-family leguminous proteinase inhibitors.     

Amino acid sequence of the acidic Kunitz-type trypsin inhibitor from winged-bean seed [Psophocarpus tetragonolobus (L.) DC]

The primary sequence of trypsin inhibitor-2 (WBTI-2) fromPsophocarpus tetragonolobus (L.) DC seeds was determined. This inhibitor consists of a single polypeptide chain of 182 amino acids, including four half-cystine residues, and an N-terminal residue of pyroglutamic acid. The sequence of WBTI-2 showed 57% identity to the basic trypsin inhibitor (WBTI-3) and 50% identity to the chymotrypsin inhibitor (WBCI) of winged bean, and 54% identity to the trypsin inhibitor DE-3 fromErythrina latissima seed. The similarity to the soybean Kunitz trypsin inhibitor (40%) and the other Kunitz-type inhibitors fromAdenanthera pavonina (30%) and wheat (26%) was much lower. Sequence comparisons indicate that thePsophocarpus andErythrina inhibitors are more closely related to each other than to other members of the Kunitz inhibitor family.     

Somatic embryogenesis and plant regeneration from leaf derived callus of winged bean [Psophocarpus tetragonolobus (L.) DC.]

Somatic embryos were obtained from callus cultures derived from leaf explants of the winged bean, Psophocarpus tetragonolobus (L.) DC. Initiation and development of the somatic embryos occurred with a two-step culture method. Callus cultures initiated on MS medium with NAA and BAP, upon transfer to a new medium with IAA and BAP, produced somatic embryos. Maximum embryogenesis of 60% was obtained on induction medium with 0.5 mg/l NAA plus 1.0 mg/l BAP followed by transfer to a secondary medium with 0.1 mg/l IAA and 2.0 mg/l BAP. Optimal embryo germination and plantlet development was achieved on MS medium with 0.2 mg/l BAP plus 0.1 mg/l IBA. The regenerated plants were successfully transferred to glasshouse conditions.Abbreviations MS Murashige and Skoog (1962) medium - 2,4-D 2,4-dichlorophenoxyacetic acid - NAA 1-naphthaleneacetic acid - IAA Indole-3-acetic acid - IBA Indole-3-butyric acid - BAP 6-benzylaminopurine - KN Kinetin     

Cross-inoculation specificity ofPsophocarpus tetragonolobus (L.) DC

Summary Only legumes of the cowpea cross-inoculation group, including the winged bean (Psophocarpus tetragonolobus) were found to form nodules in a temperate zone soil with no previous history of legume cropping. Isolates from root nodules from uninoculated winged beans grown in the field only nodulated legumes in the cowpea cross-inoculation group.Rhizobium japonicum formed ineffective nodules with the winged bean. Contribution No.5852, Scientific Article No.A2802 of the Maryland Agricultural Experiment Station, Department of Botany.     

Structure and regulated expression of Kunitz chymotrypsin inhibitor genes in winged bean [Psophocarpus tetragonolobus (L.) DC.]

We analyzed the structure and the expression of Kunitz chymotrypsin inhibitor (WCI) genes in winged bean. WCI was encoded by a multigene family which comprised at least seven members. From their primary structures, four genes (WCI-2, WCI-3a, WCI-3b, and WCI-x) were expected to be functional ones and the other three (WCI-P1, WCI-P2, and WCI-P3) to be pseudogenes. The nucleotide sequences of the WCI-3a and WCI-3b genes were nearly identical, and they encoded the WCI-3 protein, the major chymotrypsin inhibitor in seeds. The WCI-2 gene also encoded the chymotrypsin inhibitor found in seeds and the WCI-x gene was expected to encode an unidentified chymotrypsin inhibitor. WCI messenger RNA and protein accumulated mainly in developing seeds and tuberous roots, small amounts of WCI mRNA being present in stems and pericarps. In seeds, transient accumulation of WCI mRNA was observed during the seed maturation period. These results suggest that the expression of WCI genes is regulated organ-specifically and developmentally in winged bean.     

Amino acid sequence and disulfide bridges of affinity purified Kunitz-type chymotrypsin inhibitor from winged bean seed (Psophocarpus tetragonolobus (L.) DC)

The primary sequence of the affinity purified chymotrypsin inhibitor, WBCI, isolated from the albumin fraction of Psophocarpus tetragonolobus (L.) DC cv. UPS-122 seed was determined. The inhibitor consisted of a single polypeptide chain of 183 amino acids (Mr 20285) and the four half-cystine residues in the molecule formed two intramolecular disulfide bridges equivalent to those in other Kunitz-type seed inhibitors. The sequence of this chymotrypsin inhibitor was identical to that of chymotrypsin inhibitor-3 from cultivar UPS-31 and it showed about 50% sequence similarity to the winged bean acidic (WBTI-2, pI 5.1) and basic (WBTI-1, pI 8.9) trypsin inhibitors. Sequence similarities to other Kunitz-type seed inhibitors are discussed.     

Crystallization and preliminary crystallographic data of rabbit uteroglobin

Uteroglobin is a steroid-binding protein of 15,800 molecular weight, composed of two chemically identical subunits which, in the oxidized form, are covalently linked by disulphide bridges. Large crystals have been grown from ammonium sulphate solutions by vapour diffusion as well as by equilibrium dialysis. The crystals are very stable under X-rays, diffract to at least 2.2 Å resolution and belong to space group P2₁. The unit cell, with dimensions $\text{a = 43.3}\text{A}\text{?}\text{, b = 31.1}\text{A}\text{?}\text{, c = 34.5}\text{A}\text{?}\text{, and}\text{β = 90.7 °}$, contains two dimeric molecules. The crystals exhibit a prominent pseudo symmetry corresponding to space group P2₁2₁2 which indicates that the two subunits should be structurally nearly identical.     

Effect of raw winged bean [Psophocarpus tetragonolobus (L.) DC] tuber lectin on gastrointestinal tract of growing rats

The fraction containing high hemagglutinating activity was prepared from raw winged bean tubers and orally administered to growing rats. The food intake and body weights of these rats decreased as the level of lectin increased and significant lectin activity was detected in the faeces extracted from these rats which is anti-genically similar to the native lectin preparation. Microscopic examination has revealed morphological changes in the intestinal epithelial cells. The binding action of the lectin to the mucosal epithelia of the gastrointestinal tract is indicative of the deleterious effects caused by the winged bean tuber lectin.